2.1. Phage Isolation

Tomato plant samples were collected from tomato fields in Duc Trong district, Lam Dong province, Vietnam, and transported to the laboratory for phage isolation under cold conditions. Ten grams of the sample was ground and mixed with 10 mL of distilled water and 10% (w/v) chloroform. The mixture was then vortexed for 5 min before being centrifuged at 4000 rpm (Tomy MX-100, Japan) for 10 min at room temperature to obtain the supernatant and discard the plant residue and chloroform layers. To remove the remaining chloroform, the supernatant was centrifuged at 10,000 rpm for 5 min at 4°C. The resultant supernatant was filtered using a 0.45-μm pore size filter (Minisart^® NY25 17846, Sartorius, Germany), and the filtrate was used for phage enrichment. P. solanacearum PS025 which caused a disease rate of 93.3% in tomato in a 7-day pathogenicity experiment at a dose of 10⁸ CFU.mL^-1 (data not shown) was used as a host. In the initial step of phage enrichment, the filtrate was fed to a 100 μL log-phase P. solanacearum PS025 culture in a falcon tube containing 9 mL of TSB medium. The culture was centrifuged for 5 min at a temperature of 4°C after shaking at 150 rpm (LSI-3016R, Daihan Labtech, Korea) for 24 h at 30°C. The resultant supernatant was run through a 0.22-μm pore size filter (Minisart® NY25 17845, Sartorius, Germany), and the filtrate was subjected to a plaque assay. A mixture of 100 μL filtrate and 200 μL log-phase P. solanacearum PS025 culture was poured over a 1.5% Luria-Bertani (LB) agar plate. After incubating at 30°C overnight, a single transparent plaque was removed from the plate, suspended in SM buffer, incubated at 4°C overnight, and passed through a 0.22-μm filter. To purify the phage, the filtrate was subjected to the above protocol three times in a row.

2.2. Transmission Electron Microscopy Examination

A high titer (approximately 10¹⁰ PFU/mL) phage suspension sample was generated and then negatively stained with 5% uranyl acetate. Bacteriophage morphologies were examined at the Vietnam National Institute of Hygiene and Epidemiology using a transmission electron microscope (JEOL JEM-1010, Japan) operating at 80 kV voltage and an instrumental magnification of 25,000–30,000.

2.3. One-step Growth Curve

Based on previous research, the one-step growth curves of three phages were determined with minor modifications [10]. The P. solanacearum PS021 culture was incubated at 30 °C, 150 rpm until its OD₆₀₀ reached 0.1 (approximately 10⁷ CFU/mL). The phage was added at a multiplicity of infection (MOI) of 0.01. (phage: host). This mixture was incubated for 10 min at 30°C and 150 rpm before being centrifuged for 5 min at 10,000 rpm and 4°C. The pellet was resuspended in the same volume of TSB and incubated as before. Every 5 min, a portion of the volume was taken and diluted 100 fold in SM buffer on ice. The diluted samples were centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was used for phage titration using the double agar-layer technique. The phage’s latent period and burst size were measured using a previously established method [11]. The experiment was repeated three times.

2.4. In Vitro Control of Phage against P. solanacearum

The host bacterial culture in TSB was shaken at 150 rpm and 30°C until it reached an OD₆₀₀ of 0.1. Next, it was separated into four aliquots. Each of the three aliquots contained the phage at a MOI of 0.01, 0.1, or 1.0. The other aliquot contained no phage (the control). The four mixtures were shaken at 150 rpm and 30°C. The OD₆₀₀ value was periodically determined. Each trial was performed three times.

2.5. Phage Genome Sequencing and Analysis

Phage genomic DNA was extracted according to the previous study by Andrews [12]. The phage genome was utilized as input for library preparation with the NEBnext Ultra II DNA Library Prep Kit, and sequencing was done on an Illumina (NextSeq550, USA) (150 bp paired end) at KTEST (Ho Chi Minh City, Vietnam). FASTQC v0.11.9 [13] was utilized to evaluate the sequence quality of read data in FASTQ format files before further analysis. Unicycler v0.4.8 in conservative mode was utilized to carry out de novo assembly [14]. QUAST version 5.0.2 was utilized to evaluate entire contig assemblies [15]. BLAST search against the NCBI database was used to identify viral origin [16]. The GenBank files generated by RAST were then manually inspected to validate identified open reading frames. Using Proksee, a circular representation of the phage genome was generated. Using ResFinder v4.1, the presence of possible antibiotic resistance factors was explored [17]. Using BACPHILP, it was determined if the phage was more likely to be virulent (lytic) or temperate (lysogenic) [18]. tRNA detection was conducted using tRNAscan-SE v2.0 [19].

2.6. Statistical Analysis

One-way analysis of variance was used to process the resulting data using the Statistical Package for the Social Sciences version 20 software for Mac. Duncan’s multiple range test demonstrated a statistically significant difference between treatments at P ≤ 0.05.

3.1. Lytic Activity and Morphology of Phages

Three bacteriophages such as BHDT3, BHDT4, and BHDT8 were isolated from tomato plant samples obtained from Bac Hoi village, in Duc Trong district, Lam Dong province. Following purification, three phages exhibited clear plaques [Figure 1]. The phage morphologies were clarified based on transmission electron microscopy morphological analysis [Figure 1]. It was discovered that three phages have icosahedral heads with a diameter of approximately 87 nm (BHDT3), 54 nm (BHDT4), or 61 nm (BHDT8). Three phages belonged to the Podovirus family and were classified as Autographiviridae [20]. The one-step growth curves of three phages are described in Figure 2. The latent period and burst size of the BHDT3 are 205 (min) and 12 ± 1.7 (PFU/cell). The values of the BHDT4 are 235 (min) and 17 ± 2.7 (PFU/cell). The BHDT8 shows the lowest latent period of 160 (min) and the highest burst size of 45 ± 5.4 (PFU/cell). It indicated that the BHDT8 had the highest lytic activity in terms of latent period and burst size.

Figure 1: Top agar overlay showing plaque morphology of three phages BHDT3, BHDT4, and BHDT8; the scale bar indicates 1 cm. Electron micrograph of three respective phages; the bar represents 200 nm or 500 nm. [Click here to view]

Figure 2: The one-step growth curve of phage (a) BHDT3, (b) BHDT4, and (c) BHDT8. [Click here to view]

3.2. Inactivation of P. solanacearum by Phages

The capacity of three phages to inhibit P. solanacearum growth at different MOI values in TSB was evaluated. As shown in Figure 3a, four treatments experienced an increase in OD₆₀₀ during the first 2.0 h of incubation (with or without the phage). Nevertheless, from 2 h to 4 h of incubation, the OD₆₀₀ of the bacterium-phage suspension at the MOI = 1.0 began to decrease, whereas that of the bacterium-phage suspension at the MOIs = 0.1, 0.01 and the control continued to rise. From 4 h to 6 h of incubation, the OD₆₀₀ of the bacterium phage at three MOI values decreased, whereas the OD₆₀₀ of the control dramatically increased. The sharp decrease in OD₆₀₀ of the suspension was caused by the lysis of host cells by phages. The ability of BHDT3 to inactivate the host cells was demonstrated by the transparency of the bacterium-phage suspension until hour 34 (OD₆₀₀ < 0.1), whereas the turbidity of the control was maintained throughout the experiment. The OD₆₀₀ of the bacterium-phage solution increased after 34 h of incubation, indicating the growth of phage-resistant bacteria. Figure 3b and c depicted the capacity of phage BHDT4 and BHDT8 to inhibit P. solanacearum growth in TSB, respectively. The OD₆₀₀ changes at the first 4 h were similar to that shown in Figure 3a. The ability of BHDT4 to inactivate the host cells was described by the transparency of the bacterium-phage suspension until the hour 36 (OD₆₀₀ < 0.1). However, this ability of BHDT8 was much higher than that of the other two phages when the transparency of the bacterium-BHDT8 suspension was maintained until 52 h [Figure 3c]. It indicated a higher lytic activity of BHDT8 compared to BHDT3 and BHDT4. Therefore, phage BHDT8 was chosen to further analysis of its genome properties and safety for possible usage in tomato farming.

Figure 3: Changes in optical density at 600 nm (OD600) during inactivation of P. solanacearum Ps021 by (a) BHDT3, (b) BHDT4, and (c) BHDT8 in TSB at 30°C at different multiplicities of infection values of 0.01 (square), 0.1 (diamond), and 1.0 (triangle). The negative control was without phage (circles). Error bars indicating 95% confidence intervals for the averaged values (n = 3) are not graphically detectable as the intervals are too narrow. [Click here to view]

MOI value describes the number of phages against the number of host bacteria at the initial stage. In this study, the effect of three MOI values of 0.01, 0.1, and 1.0 on the inactivation of P. solanacearum by each phage was conducted and compared. There was no clear difference in the inactivation time among the three cases of MOI value. For instance, in Figure 3c, re-increase of OD₆₀₀ value occurred at the same time (52 h) at MOI values of 0.01, 0.1, and 1.0. In the application of phages to control the pathogen, it prefers a smaller MOI value since it implies a smaller required amount of phage product.

3.3. Phage Genome Analysis

The nucleotide sequences for phage BHDT8 were submitted to NCBI under accession number OQ579022. The assembly generated one complete contig, with an estimated read depth of 2051x. The phage genome was 41,298 bp long with a total GC content of 62%, containing 47 predicted protein CDSs. The whole genome of phage BHDT8 encoding a variety of different functional proteins was annotated [Figure 4]. Genes related to DNA replication and modification enzymes were detected such as polymerase, exonuclease, endonuclease, helicase, primase, and peptidoglycan lytic exotransglycosylase. Furthermore, one lysozyme-encoding gene was also found in this genome. Genes encoding structural and packaging proteins were detected including capsid protein, head-to-tail connector, tail fiber, and tail tubular. The remaining predicted proteins were categorized as hypothetical [Supplementary Data from Table S1].

Figure 4: Genome map of BHDT8. Relative orientations of open reading frames are indicated blue colors (sense and antisense). [Click here to view]

The lifestyle of phage can be categorized into two states, lytic or lysogenic, with the former being essential for phage therapy candidates [21]. In its lysogenic state, a phage integrates its DNA into the host’s DNA to become a prophage, rendering itself dormant and suppressing the anti-bacterial properties typically displayed during its lytic state. The presence of a functional phage-encoded enzyme, integrase, is largely responsible for lysogeny’s mechanisms. Therefore, the presence of an integrase gene is not accepted for the therapy. Detecting any of the following would likely prevent a phage for therapeutic use: Genes encoding virulence factors, antimicrobial resistance, toxins, and transducing elements. Therefore, before using phages to control P. solanacearum infections in tomato, it is necessary to conduct a comprehensive genomic characterization of the phages. In this study, no known antibiotic resistant genes or virulence factors of pathogenic bacteria were detected in phage BHDT8. In addition, no known genes associated with lysogeny, such as integrase, were identified. The lifestyle prediction of the phage indicated that BHDT8 was likely to be virulent. The phage’s lytic nature suggested that it could serve as a potential agent against P. solanacearum, an infectious agent in Vietnam’s tomato.